Oct 16, 2023

Public workspaceMicroscopy-based GSH bead protein-protein interaction assay

DOI
dx.doi.org/10.17504/protocols.io.4r3l27xdxg1y/v1
  • 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Minghao Chen
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DOI: dx.doi.org/10.17504/protocols.io.4r3l27xdxg1y/v1
Protocol Citation: Minghao Chen, Xuefeng Ren 2023. Microscopy-based GSH bead protein-protein interaction assay. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l27xdxg1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 05, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82919
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-000350
Abstract
Microscopy-based GSH bead protein-protein interaction assay
Wash pre-blocked glutathione sepharose beads (GE Healthcare) with the reaction buffer (25 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM MgCl2 and 1 mM TCEP) three times.
Make a mixture of 1 μM purified GST tagged protein and 500 nM purified fluorescent protein in total 70 μl
Incubate at TemperatureRoom temperature for Duration00:30:00 , samples were mixed with additional 100 μL reaction buffer.

30m
Transferred to the observation chamber for imaging.
Acquire images on a Nikon A1 confocal microscope with a Nikon Plan APO VC 20x/0.75 NA UV Microscope Objective.